Project Summary: Emerging and zoonotic infectious diseases (EZIDs) cause global health devastation and are increasing in prev- alence due to human activity and other factors that facilitate disease spread. Often, the etiological agents of EZIDs are difficult to study due to lack of established tools and genetic systems to perform hypothesis-driven research. The objective of this proposal is to use bacteria of the genus Legionella as model pathogens to char- acterize host defense mechanisms applicable to EZIDs. Legionella species are natural pathogens of protozoa and accidental human pathogens that can cause disease upon inhalation of contaminated aerosols and subse- quent bacterial replication within alveolar macrophages. As transmission between mammals is rare, Legionella has not acquired sophisticated immune evasion mechanisms, and are, therefore, excellent model pathogens to reveal host immune defense mechanisms. Legionella replication within phagocytic cells is facilitated by a Dot/Icm type IV secretion system (T4SS), which translocates a repertoire of bacterial proteins called effectors into in- fected host cells. Although effector translocation is required for intracellular replication, effector functions can also impair Legionella fitness in mammals. The overall objective is to elucidate mechanisms by which effector function contributes to pathogen clearance by the innate immune system and determine whether these can be used to enhance defense against other pathogens. The central hypothesis is that LegC4 interacts with host factors to enhance cytokine-mediated host defense. To test the central hypothesis, we will test the following specific aims. Aim 1 is to define the mechanism of LegC4-mediated attenuation of bacterial replication in mac- rophages; Aim 2 is to identify host factors modulated by LegC4; and Aim 3 is to determine if LegC4 is able to protect mice from infection with a highly virulent Legionella species that does not naturally encode LegC4. Through this work, we will elucidate the function of the effector LegC4 and further determine if LegC4 is sufficient to promote host defense against non-pneumophila pathogens. To address these questions, we will use cell biology, imaging, immunology, biochemistry, genetic techniques and animal models. The proposed project is innovative and has potential to positively impact public health. Mechanisms by which the mammalian immune system detects and eradicates pathogens can be harnessed to treat and prevent infectious diseases. The ben- efits of studying innate immune activation by effectors are (1) previously uncharacterized pathogen detection strategies may be identified and targeted for therapeutic intervention; and (2) the effectors can be used as ther- apeutics to enhance immune clearance of pathogenic microbes. Ultimately, the work will culminate in enhanced understanding of host defense strategies and provide the means to develop therapeutics that will be effective against a broad range of infectious agents. This is especially important to limit disease outbreaks and decrease the global health burden associated with EZIDs. This work will provide preliminary data required for a successful R01 application to investigate mechanisms of effector-mediated host immune restriction of bacterial pathogens.